Revision as of 20:43, 24 July 2012

Contents

Overview

Whole plasmid PCR protocol using PFU Ultra DNA polymerase. PFU ligase is used in the reaction to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to repair the nicks.

Materials

For a 50 μL whole plasmid PCR PCR reaction:

35 μL H2O

5 μL 10X PFU Ultra PCR buffer

5 μL 2mM (each) dNTP mix

1 μL 5nM plasmid template (0.1 nM final)

1 μL 10μM sense primer (200nM final)

1 μL 10μM antisense primer (200nM final)

1 μL PFU Ultra II high-fidelity DNA polyermerase

1 μL PFU ligase

Procedure

In a PCR tube, mix the components on ice in the order they are listed above.